Glioblastoma (GBM) is the most common malignant brain tumor in human with extremely poor prognosis. Novel treatment strategies to this deadly disease are desperately needed. Cancer stem-like cells (CSLCs) have been prospectively isolated from GBM and shown required for tumor propagation. However, it has been shown that GBM CSLCs reside within niches (including endothelial niche) and are resistant to chemo- and radiation-therapy. Understanding the molecular communication between niche cells and CSLCs will help develop novel therapeutic strategies for GBM patients based on targeting both CSLCs and their niches. Our long-term goal is to develop novel therapeutic strategies for GBM patients through investigating the molecular mechanism by which signaling pathways and tumor microenvironment regulate CSLCs in GBM. We and others have demonstrated recently that Notch pathway blockade with a gamma-secretase inhibitor (GSI) depletes GBM CSLCs, inhibits tumor growth, and prolongs survival of mice bearing intracranial xenografts. The overall objective of this application is to define if acquired Notch activity in GBM CSLCs comes from Notch ligands expressed in endothelium and differentiated tumor cells (niche cells), to study the molecular mechanism by which Notch signaling regulates GBM CSLCs within their niches, and to investigate if targeting both GBM CSLCs and niche cells can improve the treatment of GBM in a pre-clinical model. In specific Aim1, we will examine if Notch ligands expressed in niche cells (endothelial cells and differentiated tumor cells) contribute to the activation of Notch in GBM CSLCs. We will first identify which Notch ligands are expressed in endothelial cells and differentiated tumor cells in primary GBM samples. Then, we will examine if knockdown of Notch ligands in niche cells will reduce growth of co-cultured GBM CSLCs in vitro and in vivo. In specific Aim2, we will define the molecular mechanism by which ligand-induced Notch activation regulates GBM CSLCs. We will use both gain and loss of Notch-function studies to identify the targets that mediate Notch-regulated proliferation, apoptosis, and differentiation in GBM CSLCs. In specific Aim3, we will examine if targeting both CSLCs and their niche will improve the treatment of GBM in a pre-clinical mouse model. We will examine if a combination targeting GBM CSLCs by GSI and targeting endothelial cell niche by VEGF inhibitor (Avastin) will improve survival of mice bearing intracranial xenografts derived from human primary GBMs. Success in the current proposal will not only enhance our understanding the molecular communication between GBM CSLCs and their niche, but will also help develop novel therapies for GBM patients based on targeting both CSLCs and their niches. Although the current research focuses on GBM, such results will also have general implications for multiple forms of neoplasia.